Ethylenediamine-disuccinate (EDDS, also referred to as ethylenediamine disuccinic acid) is a hexadentate chelating agent. EDDS has two stereo centers and, accordingly, presents three different stereoisomers, namely [R,R]-EDDS, [S,S]-EDDS and [R,S]-(meso)-EDDS. It has proved that exclusively the [S,S]-EDDS stereoisomer is subject to complete biological degradation (Schowanek et al., Chemosphere (1997), 34(11):2375-91).
Also, EDDS is a structural isomer of ethylenediamine-tetraacetate (EDTA), a widely-used and likewise hexadentate chelating agent. Both compounds are very similar in their chemical characteristics, in particular in view of their capability of chelating metal ions. Thus, EDDS and EDTA exhibit comparable chelating constants for quite a variety of metal ions.
For many decades, EDTA has been employed for removal of metal ions in most different fields on account of its pronounced chelating capability. At present, it is the most used chelating agent. Particularly stable complexes are formed with copper(II), nickel(II), iron(III) and cobalt(II) ions, but also with heavy metal ions and calcium and magnesium ions.
Therefore, EDTA is in particular added to detergents as a water softener, but is also used to stabilize bleaching liquors in the field of paper and textile industry, and is applied as a fertilizer in the form of iron, copper and zinc complexes thereof. Likewise, EDTA is employed in the medical field to treat heavy metal intoxication.
A drawback is that EDTA is not biodegradable and, thus, may be detected in ubiquitous waters. EDTA is considered to be eco-unfriendly, in particular due to the fact that it can dissolve heavy metals from sediments and make them bioavailable in this way.
With this background it is desirable to replace EDTA by equivalent, however, biologically degradable compounds in terms of sustainable material policy.
EDDS in the form of the biodegradable stereoisomer [S,S]-EDDS represents a generally utile alternative material owing to chelating constants comparable to EDTA.
The chemical synthesis of [S,S]-EDDS starting from L-aspartic acid and 1,2-dibromomethane in the presence of trivalent cobalt is well-known (Neal and Rose, Inorganic Chemistry (1968), 7(11):2405-12). A drawback thereby is the toxic side product hydrogen bromide which needs extensive removal. Moreover, the synthesis is done using fossil educts.
Furthermore, a non-enantioselective chemical method is well-known, wherein maleic acid or maleic anhydride and ethylene diamine are reacted, producing a racemic mixture of [R,R]-EDDS and [S,S]-EDDS together with 50% meso-EDDS. However, due to the low yield of [S,S]-EDDS and the basically very laborious racemate separation, the method is generally inappropriate for industrial application.
A biocatalytic method of producing [S,S]-EDDS is disclosed in EP 0 731 171 A2. Using the procedure described therein, [S,S]-EDDS can be obtained starting from fumaric acid and ethylene diamine under the action of microorganisms exhibiting lysis activity in an optical purity of up to 97%. Another biocatalytic method of producing [S,S]-EDDS is disclosed in EP 1 043 400 A1, wherein [S,S]-EDDS can be obtained starting from maleic acid and ethylene diamine in the presence of microorganisms exhibiting maleate isomerase activity and metal ions in an optical purity of up to 98%. However, both the biocatalytic methods rely on the use of synthetic precursors that cannot be provided by the microorganisms themselves.
Furthermore well-known is the biosynthesis of [S,S]-EDDS using the bacteria species Amycolatopsis japonicum (Zwicker et al., Journal of Industrial Microbiology & Biotechnology (1997); 19(4):280-285). However, a drawback thereby is that the biosynthesis is zinc-dependent, and a zinc concentration as low as 2 μM in the culture medium is capable of causing an almost complete disruption of the [S,S]-EDDS synthesis (Cebulla I., Thesis (1995), University of Tubingen).
A method using zinc-free reaction conditions to produce [S,S]-EDDS by Amycolatopsis japonicum and using an optimized culture medium is described in WO 96/36725 A1.
In particular the fact that application of the synthesis procedures in a large scale has proved to be complicated and uneconomical due to the zinc dependency and low yields related thereto, is an issue with the generic biosynthesis methods for [S,S]-EDDS. Indeed, generating a zinc-free environment in culture media and fermenters entails considerable expenses and is almost impossible.
With this background, it could therefore be helpful to provide proteins or peptides, nucleic acids, gene clusters, vectors, host cells, bacterial cells, and a method and a kit for the biosynthesis of [S,S]-EDDS.